Campylobacter jejuni, an important foodborne pathogen causing gastroenteritis in humans, has evolved multiple mechanisms to counteract the action of various antibiotics, which has posed a serious threat to public health, Many of these resistance mechanisms, such as gyrA mutations and beta-lactamase production, confer Campylobacter resistance to specific antibiotics. However, the active efflux systems, which extrude structurally diverse antibiotics out of bacterial cells, contribute to the intrinsic and acquired resistance to multiple drugs. Although previous studies suggested the possible presence of functional efflux systems m C. jejuni, the antibiotic efflux machinery in this pathogen has not been defined. Using transposon mutagenesis in conjunction with other approaches, we have recently characterized a three-gene operon (named cmeABC) encoding a tripartite antibiotic efflux pump that contributes to C. jejuni resistance to structurally unrelated antibiotics, heavy metals, bile salts, and other toxic compounds. Our preliminary data and the genomic sequence of C. jejuni NCTC 11168 suggested the presence of an additional antibiotic efflux system (name cmeDEF) and the possible regulation of cmeABC and cmeDEF by transcriptional repressors. Based on these observations and the known features of bacterial antibiotic efflux systems, we hypothesize that CmeDEF in conjunction with CmeABC plays an important role in extruding various agents, and the modulated expression of the efflux pumps by regulatory proteins contributes significantly to the intrinsic and acquired resistance of Campylobacter to multiple antimicrobials. To test our hypothesis, we plan to i) determine the role of CmeDEF and its interplay with CmeABC in mediating Campylobacter resistance to multiple drugs and ii) to identify and characterize the transcriptional repressors that modulate the expression of the antibiotic efflux systems. Various genetic and biochemical approaches, including random and site-specific mutagenesis, recombinant proteins, substrate accumulation assay, and DNA binding assays will be utilized to define the functions of the efflux systems and their interplay with regulatory proteins. It is anticipated that the proposed studies will close a major gap in our understanding of the antibiotic resistance mechanisms in C. jejuni and may open new avenues for the design of effective means to prevent and treat antibiotics-resistant Campylobacter.